This invention pertains to the location of a human glucocorticoid receptor gene promoter region and to three splice variants of the human glucocorticoid receptor gene; and the use of the promoter region and of the splice variants to improve the diagnosis and treatment of leukemia.
The use of naturally occurring substances, such as hormones, to treat cancer has certain advantages. Although side effects can occur, the effects are usually less severe than those caused by cytotoxic chemotherapy. Unfortunately most cancers are not effectively controlled by hormonal therapy, but exceptions include certain hormonally-dependent breast cancers that can be treated with the anti-estrogen tamoxifen, and acute promyelocytic leukemia that is responsive to all-trans retinoic acid. Additionally, some lymphoid malignancies can be effectively treated with glucocorticoid steroid hormones, hormones that control a variety of metabolic and developmental processes. See R. R. Denton et al., xe2x80x9cDifferential Autoregulation of Glucocorticoid Receptor Expression in Human T- and B-Cell Lines,xe2x80x9d Endocrinology, vol. 133, pp. 248-256 (1993). Certain types of B- and T-cell acute lymphoblastic leukemia (xe2x80x9cALLxe2x80x9d) are particularly sensitive to glucocorticoid hormonal therapy. Glucocorticoids affect lymphoid malignancies due to the induction of programmed cell death, or apoptosis, of immature lymphocytes. See C. W. Distelhorst, xe2x80x9cBasic and Clinical Studies of Glucocorticosteroid Receptors in Lymphoid Malignancies,xe2x80x9d pp. 494-515 in W. V. Vedeckis (ed.) Hormones and Cancer (1996).
The cytolytic effect of glucocorticoids is mediated by the glucocorticoid receptor (GR). Upon entering the cell, glucocorticoids bind to the soluble intracellular receptor protein GR, causing an alteration in GR structure. This alteration in structure converts the unactivated receptor to the activated form that both binds to specific DNA sequences and facilitates transcription of glucocorticoid-responsive genes. The transcribed glucocorticoid-induced mRNA messages are then transported into the cytoplasm and translated into specific proteins. The changes in concentration and types of the intracellular proteins modulate a variety of intracellular processes. GR concentration has been shown to correlate with sensitivity to steroid treatment in vitro. See J. N. Vanderbilt et al., xe2x80x9cIntracellular Receptor Concentration Limits Glucocorticoid-Dependent Enhancer Activity,xe2x80x9d Mol. Endocrinol., vol. 1, pp. 68-74(1987). Additionally, an in vivo study of large numbers of patients with ALL found that a low GR level in lymphoblasts isolated at the initial diagnosis was significantly correlated with a poor response to therapy, shorter duration of remission, and a poor overall prognosis. Distelhorst (1996).
Studies have shown that chronic glucocorticoid treatment of cells that do not respond by apoptosis resulted in a decrease in expression (or down-regulation) of the GR gene, as evidenced by decreased levels of GR mRNA and protein. See S. Okret et al., xe2x80x9cDown-Regulation of Glucocorticoid Receptor mRNA by Glucocorticoid Hormones and Recognition by the Receptor of a Specific Binding Sequence Within a Receptor cDNA Clone,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 83, pp. 5899-5903 (1986); and Dong et al., xe2x80x9cRegulation of Glucocorticoid Receptor Expression: Evidence for Transcriptional and Posttranslational Mechanisms,xe2x80x9d Mol. Endocrinol., vol. 2, pp. 1256-1264 (1988). By contrast, in cells that undergo apoptosis upon glucocorticoid treatment, such as immature thymocytes, T-lymphocytes, and leukemic T-lymphoblasts, glucocorticoid treatment caused a dramatic increase in the levels of GR mRNA and protein levels, indicating an increase in expression (or up-regulation) of the GR gene. Denton et al. (1993). The molecular mechanisms that control down-regulation and up-regulation of the GR gene are not understood.
Studies on the structure of the human GR gene have shown that the mature GR mRNA is coded by nine separate exons in the genomic DNA. See I. J. Encio and S. D. Detera-Wadleigh, xe2x80x9cThe Genomic Structure of the Human Glucocorticoid Receptor,xe2x80x9d J. Biol. Chem., Vol. 266, pp. 7182-7188 (1991). Exon 1 is an untranslated exon; i.e., it is transcribed into mRNA but does not code for amino acids in the GR protein. Exon 2 contains the ATG methionine initiator for protein translation. Thus any exon 1 sequences that are spliced onto exon 2 will have no effect on the amino acid sequence of the GR, because the protein coding sequence begins in exon 2, and also because there is an in-frame stop codon located three codons upstream of the initiator ATG in exon 2. Exon 9 codes a long 3xe2x80x2 untranslated region in the GR mRNA that contains two potential polyadenylation addition sites. The two major, mature GR mRNA species are about 7 kilobases and 5 kilobases long, depending upon which polyadenylation site is used. Additionally, an alternative splicing event in exon 9 gives rise to two GR protein forms, GR alpha and GR beta. GR alpha is the major, functional species found in all cell types. The function, if any, of GR beta is not clear.
The GR promoter region, which controls GR gene expression and mRNA synthesis, has been investigated in both the human and mouse. The human GR promoter was found to be xcx9c2.7 kilobase pairs (kbp). See J. Zong et al. xe2x80x9cThe Promoter and First, Untranslated Exon of the Human Glucocorticoid Receptor Gene are GC Rich but Lack Consensus Glucocorticoid Receptor Element Sites,xe2x80x9d Mol. Cell. Biol., vol. 10, pp. 5580-5585 (1990) and Y. Nobukuni et al., xe2x80x9cCharacterization of the Human Glucocorticoid Promoter,xe2x80x9d Biochemistry, vol. 34, pp. 8207-8214 (1995). This GR promoter is GC-rich and lacks a TATA box and CAAT box, common characteristics of xe2x80x9chousekeepingxe2x80x9d genes that are constitutively expressed in most cell types.
The GR protein is found in virtually all cells in the human body. Regulatory elements that control GR gene expression have been characterized in the 2.7 kbp human GR promoter region,just upstream from the untranslated exon 1. See Nobukuni et al. (1995); M. B. Breslin and W. V. Vedeckis, xe2x80x9cThe Glucocorticoid Receptor and c-jun Promoters Contain AP-1 Sites that Bind Different AP-1 Transcription Factors,xe2x80x9d Endocrine, vol. 5, pp. 15-22 (1996), P. Wei and W. V. Vedeckis, xe2x80x9cRegulation of the Glucocorticoid Receptor Gene by the AP-1 Transcription Factor,xe2x80x9d Endocrine, vol. 7, pp.303-310(1997), and M. B. Breslin and W. V. Vedeckis, xe2x80x9cThe Human Glucocorticoid Receptor Promoter Upstream Sequences Contain Binding Sites for the Ubiquitous Transcription Factor, Yin Yang 1,xe2x80x9d J. Steroid Biochem. Molec. Biol., vol. 67, pp. 369-381 (1998). Eleven regions in the first 800 bp of the promoter have been identified that bind protein. Of these eleven, three regions bind the transcription factor Sp1, two regions bind Sp1 and another protein, and one region binds predominantly the transcription factor AP-2, as well as some Sp1. The binding of Sp1 to GC-rich regions is typical of housekeeping, constitutive promoters. See Nobukuni et al. (1995). A single untranslated exon in the 2.7 kbp human GR promoter gene fragment has also been identified. See Zong et al., (1990) and I. J. Encio and Detera-Wadleigh (1991). The start sites for transcription that were identified in the human GR promoter region were somewhat variable, which is another characteristic of a promoter for a housekeeping gene.
The mouse GR promoter and gene structure have also been characterized. See Strxc3xa4hle et al., xe2x80x9cAt Least Three Promoters Direct Expression of the Mouse Glucocorticoid Receptor Gene,xe2x80x9d Proc. Natl. Acad. Sci. USA., vol. 89, pp. 6731-6735 (1992). Similar to human, the mouse GR transcript derives from 9 exons, and the ATG methionine initiator is in exon 2. However, two exon 1 sequences that derive from the mouse promoter analogous to the human 2.7 kpb sequence have been identified. The one nearest to exon 2 has been designated exon 1C, and is homologous to the human GR exon 1 sequence. See Nobukuni et al., 1995. Further upstream is untranslated exon 1B, found about 1 kbp upstream from the exon 1C sequence. An additional untranslated exon (1A) was found on some GR transcripts. The genomic sequence that coded for exon 1A was located far upstream (xcx9c32 kbp) from the mouse GR promoter sequence that gives rise to the exon 1B and 1C GR transcripts. Thus mouse GR mRNA transcripts derive from three separate promoters, which give rise to three GR transcripts that differ in the untranslated exon 1 region (1A, 1B, or 1C). Significantly, exon 1B- and 1C-containing mouse GR transcripts were detected in all tissues and cells studied, namely brain, liver, fibroblast, and T-lymphoma cell lines. However, exon 1A-containing transcripts were only found in two T-cell lymphoma cell lines, leading to the conclusion that the mouse 1A promoter was cell-specific for T lymphocytes. There is substantial sequence homology between the 2.7 kbp human GR promoter and that of mouse exons 1B and 1C.
Other alternative transcripts for the mouse GR gene have recently been described. See Chen et al., xe2x80x9cMultiple Glucocorticoid Receptor Transcripts in Membrane Glucocorticoid Receptor-Enriched S-49 Mouse Lymphoma Cells,xe2x80x9d J. Cell. Biochem., vol. 74, pp. 418-429 (1999a). Both of the previously-described exons 1A and 1B (but not exon 1C) were found in GR transcripts in the mouse S-49 lymphoma cell line. Two new untranslated exon 1-containing mouse GR transcripts were also discovered (exons 1D and 1E). Exon 1D genome sequences are located xcx9c300 bp upstream of the exon 1B region. Exon 1E sequences overlap with exon 1C sequences, but contain sequences not found in exon 1C. The exon 1A-containing transcript was present in high amounts in cells that were enriched for a membrane-associated form of the GR, compared to cells that had low levels of membrane GR, e.g., S-49 cells selected for low membrane GR levels and mouse AtT-20 pituitary tumor cell line.
The mouse GR promoter and exon 1A sequences have been characterized. See Chen et al, xe2x80x9cAssociation of the Glucocorticoid Receptor Alternatively-Spliced Transcript 1A with the Presence of the High Molecular Weight Membrane Glucocorticoid Receptor in Mouse Lymphoma Cells,xe2x80x9d J. Cell. Biochem., vol. 74, pp. 430-446 (1999b). Using a variety of techniques, a new exon (termed A), based on the nomenclature of Strxc3xa4hle et al. (1992), was identified. Exon 1A is 1013 bp in length. Transcripts that contain exon 1A were suggested to result in the synthesis of a larger, membrane-associated GR, whose presence correlates with apoptosis in a mouse lymphoma cell line. Only one exon 1A-containing mouse GR transcript was identified. A putative promoter region of the 1A-containing transcripts was postulated. This was based upon the fact that the sequences (2140 bp) upstream of the identified start site of exon 1A were not found to be transcribed into RNA in the cell. Computer analysis indicated possible transcription factor binding sites that might be consistent with this being a promoter region. Transfection of the full-length exon 1A-containing GR cDNA into mouse AtT-20 pituitary cells and human HL-60 myeloid leukemia cells, followed by hormone treatment, resulted in cell death. These studies suggested that the exon 1A-containing mouse GR mRNA was a necessary factor hormone-induced cell death, since the steroid hormone alone does not normally kill these two cell lines.
A mechanism used to generate different protein products, is alternative RNA splicing of transcripts that are controlled by the same promoter. Alternative splicing of a precursor to different mature mRNA transcripts is a complicated process. A variety of small nuclear RNA""s and proteins form complexes with the pre-mRNA, and splicing involves specific sequences at the splice donor site, splice acceptor site, and the branch point. P. A. Sharp, xe2x80x9cSplit Genes and RNA Splicing,xe2x80x9d Cell, vol. 77, pp. 805-815 (1994). In some cases, as for the calcitonin/calcitonin-related peptide system, alternative splicing of protein-coding exons gives rise to different protein products with different biological functions. S. G. Amara et al., xe2x80x9cAlternative RNA Processing in Calcitonin Gene Expression Generates mRNAs Encoding Different Polypeptide Products,xe2x80x9d Nature, vol. 298, pp.240-244 (1982). V. Jonas et al., Alternative RNA Processing Events in the Human Calcitonin/Calcitonin Gene-Related Peptide Gene Expression, xe2x80x9cProc. Natl. Acad. Sci. USA, vol. 82, pp. 1994-1998 (1985). Differential mRNA splicing can occur in a cell type- and developmental stage-specific manner, and can be regulated by both positively-acting (stimulatory) and negatively-acting (repressive) cell specific protein factors. P. J. Grabowski, xe2x80x9cSplicing Regulation in Neurons: Tinkering with Cell-Specific Control,xe2x80x9d Cell, vol. 92, pp. 709-712 (1998).
In a clinical setting, there is a need for a method for determining the GR status of a patient to help design an effective therapeutic strategy. A patient with lymphoid malignancies such as ALL frequently develops glucocorticoid resistance, which leads to a relapse even though initial glucocorticoid treatment resulted in the killing of leukemic cells and a remission. See Distelhorst (1996). Thus, although relapsed ALL patients are non-responsive to glucocorticoid treatment, they are still at risk for developing the side-effects of corticosteroid therapy, including hypertension, hyperglycemia, and immunosuppression. In addition, the continued glucocorticoid treatment of a glucocorticoid-resistant patient may preclude the use of more aggressive chemotherapy due to the patient""s poor condition caused by the side-effects of corticosteroid therapy. Thus, there exists a need to be able to identify glucocorticoid-resistant patients, in both newly diagnosed and relapsed ALL patients.
We have discovered a new sequence, hGR 1Ap/e, isolated from human DNA upstream from the previously known 2.7 kbp human GR promoter region. This new sequence was found to contain a new promoter (the human 1A GR promoter) and a new untranslated exon sequence (human GR exon 1A) that occurs in the mRNA that also contains the sequence that codes for the human glucocorticoid receptor protein (hGR). The hGR 1Ap/e sequence is approximately 25 kilobase pairs upstream of the hGR coding sequence. Alternative splicing produces three different hGR 1A-containing transcripts, 1A1, 1A2, and 1A3. GR transcripts containing exons 1A1, 1A2, 1B (in the proximal promoter region), and 1C (in the proximal promoter region) are expressed at various levels in many cancer cells and in human brain. Exon 1A3-containing GR transcripts appear to be restricted to blood cell cancers and to human brain. Glucocorticoid hormone treatment caused an up-regulation of exon 1A3-containing GR transcripts in T-lymphoblast cells, and a down-regulation of exon 1A3-containing transcripts in B-lymphoblast cells. These alterations correlate with the known responses of the two cells to glucocorticoid hormone treatment, i.e., B-lymphoblast cells are known to be resistant to glucocorticoid hormone treatment and T-lymphoblast cells are known to be sensitive. Thus the presence of exon 1A3 -containing transcripts can be used to detect cancerous blood cells that would be sensitive to glucocorticoid hormone treatment. Additionally, an interferon regulatory factor element (IRF-E) that binds IRF-2 was found in the exon 1A sequence. This regulatory factor appears to contribute significantly to basal transcription rate of 1A GR transcripts. The intraexonic location of this sequence was surprising. A glucocorticoid response element (GRE) was also found intraexonically in the exon 1A sequence. The presence of these two regulatory factors indicates that both interferon and glucocorticoid hormone could be used to increase the level of exon 1A3-containing transcripts in the cells. There are xcx9c1075 base pairs of hGR 1A promoter sequence, based upon the absence of these sequences in mRNA. There are xcx9c981 bp of exon 1A sequence. The portions of the hGR 1Ap/e sequence that can function as a eukaryotic promoter or as intraexonic regions that influence promoter activity were identified based on reporter gene assays. The detection of exon 1A3-containing transcripts can be used for the diagnosis of patients with T-cell acute lymphoblastic leukemia (ALL) and other glucocorticoid-responsive cancers, and to identify patients who would benefit from glucocorticoid hormone treatment.